Human Cytomegalovirus (HCMV) is a leading cause of birth defects. Ramifications of HCMV infection are primarily observed in the central nervous system (CNS) and include hearing loss, vision loss, microcephaly and mental retardation. Despite considerable effort, the underlying mechanisms causing these CNS defects remain unknown. Our previously funded studies of HCMV interaction with the host cell DNA and its DNA repair machinery have identified three areas of particular interest to pursue. First, HCMV is one of only two viruses known to inflict site-specific chromosomal damage to the host DNA. Fine mapping of the chromosome 1q breaksites has revealed two genes, nidogen 1 (NID1) and myelin protein zero (MPZ), linked to the development of hearing loss in infected infants. Second, studies on DNA repair in HCMV infected permissive fibroblasts found that, although DNA damage responses were activated during infection, they were not completed. We suspect that compromised repair of specific and nonspecific DNA damage may play a role in the development of HCMV-induced birth defects. Third, we have recently begun working with a promising new in vitro model, the Neural Progenitor Cell (NPCs), and its derivatives. These cells, derived from post mortem neonatal brain tissue, provide a unique opportunity to investigate HCMV infection in a model system directly relevant to the human fetal CNS. NPCs, their glial derivatives, and the large majority of their neuronal derivatives, are fully permissive and suffer a lytic infection. However, a subpopulation of differentiated neurons, although permissive, exhibit extended viral antigen expression and release of virions. The long term goal of our work is to translate the information gained from studying infection in vitro, into understanding the development of CNS defects in congenitally infected infants. We propose examining clinical specimens for confirmatory evidence of the results found in our in vitro experiments. We have procured sample archival brain and auditory system tissues from neonates that have succumbed to HCMV infection, which will feature prominently in our proposed experiments. We will advance our long term goal with the testing of four hypotheses: 1) that a viral protein (or proteins) induces the site-specific breaks on Chromosome 1q; 2) that the compromised repair of HCMV-induced breaks causes downregulation of breaksite-encoded genes; 3) that HCMV disrupts the cellular DNA repair machinery's ability to repair non-specific damage in neural cells; and 4) that HCMV infection within the CNS affects expression of specific genes involved in differentiation, migration and cell function in NPCs and long-term neurons. The experiments described in this proposal will provide a detailed understanding of the molecular mechanisms underlying the genesis of HCMV-induced birth defects and contribute to the development of strategies to interrupt these mechanisms and, hopefully, prevent their frequently devastating consequences.